Synergy between an emerging monopartite begomovirus and a DNA-B component

In recent decades, a legion of monopartite begomoviruses transmitted by the whitefly Bemisia tabaci has emerged as serious threats to vegetable crops in Africa. Recent studies in Burkina Faso (West Africa) reported the predominance of pepper yellow vein Mali virus (PepYVMLV) and its frequent association with a previously unknown DNA-B component. To understand the role of this DNA-B component in the emergence of PepYVMLV, we assessed biological traits related to virulence, virus accumulation, location in the tissue and transmission. We demonstrate that the DNA-B component is not required for systemic movement and symptom development of PepYVMLV (non-strict association), but that its association produces more severe symptoms including growth arrest and plant death. The increased virulence is associated with a higher viral DNA accumulation in plant tissues, an increase in the number of contaminated nuclei of the phloem parenchyma and in the transmission rate by B. tabaci. Our results suggest that the association of a DNA-B component with the otherwise monopartite PepYVMLV is a key factor of its emergence.

www.nature.com/scientificreports/ referred to as the common region (CR), contains the origin of replication: a conserved hairpin structure, with an upstream Rep binding iteron sequence 3,10 . The similarity of the CRs ensures binding between the DNA-Aencoded Rep and the cognate DNA-B. Furthermore, in natura CR recombinations have been described between DNA-A & -B components 11 . Since 1980s, begomoviruses have emerged in many areas of the world and extensively described in tomato (Solanum lycopersicum) 12 , they have become a major constraint in the production of vegetables. In Africa, a complex of at least 20 monopartite begomovirus species are involved in tomato yellow leaf curl or tomato leaf curl diseases (TYLCD and ToLCD), including seven species described in West Africa 13,14 . Among them, the species Pepper yellow vein Mali virus has been identified as the most prevalent and severe tomato-infecting begomovirus in tomato and pepper in Burkina Faso 15 . Interestingly, whereas pepper yellow vein Mali virus (PepYVMLV) was originally described as a Western African monopartite begomovirus 16,17 , it has frequently been found associated with a DNA-B component 15 . The vast majority of the components of tomato-infecting bipartite begomoviruses have an obligate relationship 18,19 . However, this obligate relationship appears to be absent for both tomato yellow leaf curl Thailand virus (TYLCTHV) and tomato leaf curl Gujarat virus (ToLCGV) DNA-A components because they are able to induce systemic and symptomatic infections in the host plants, Nicotiana benthamiana 20 and in tomato 21 , respectively, in the absence of their cognate DNA-B. These data suggest that these viruses represent evolutionary intermediates between monopartite and bipartite begomoviruses.
To understand the contribution of the DNA-B component to the biology of the otherwise monopartite PepYVMLV, we evaluated biological traits related to their virulence, virus accumulation and their location in plant cellular tissue, as well as transmission of the virus by mechanical-, agrobacterium-and whitefly-mediated inoculation. We demonstrated that even though the DNA-B component is not essential for infection, it increases viral accumulation and the number of infected nuclei, the virulence and the transmission rate of PepYVMLV by Bemisia tabaci. Taken together, our results suggest that the recruitment of a DNA-B component by the monopartite PepYVMLV is a key epidemiological factor that has enabled PepYVMLV to become the most prevalent virus responsible for the most severe viral disease of tomato crops in Burkina Faso.

Higher infectivity and acute virulence of PepYVMLV DNA-A associated with DNA-B. N.
benthamiana and S. lycopersicum (tomato) plants agro-inoculated with PepYVMLV DNA-A alone or associated with DNA-B developed strikingly distinct symptoms of leaf crumpling with yellowing and stunting (Fig. 1a,b). N. benthamiana plants agro-inoculated with PepYVMLV DNA-A (of which 92.5% tested positive for DNA-A using conventional PCR) developed mild symptoms in 82.5% of inoculated plants (Table 1, Fig. 1a). In contrast, in mixed agroinoculation with DNA-B, very severe symptoms were observed in N. benthamiana (of which 100% tested PCR-positive for DNA-A & B) with plant growth arrest in 85% of inoculated plants. When tomato plants were agro-inoculated with PepYVMLV DNA-A, 20% developed very mild symptoms (Table 1, Fig. 1b), even though viral DNA-A was detected by PCR in 80% of inoculated plants. In contrast, in mixed agroinoculation with DNA-B, very severe symptoms were observed in tomato plants (of which 87% tested PCR-positive for DNA-A & B), with growth arrest in 95% and death in 17% of inoculated plants (Table 1, Fig. 1b). As positive control, tomato plants inoculated with the highly infectious TYLCV-IL DNA-A in single or mixed infection with DNA-B (of which 100% tested PCR-positive for TYLCV-IL) developed typical symptoms of TYLCV disease (Table 1). Interestingly, DNA-B was detected by PCR in 28% of mixed agroinoculated tomato plants with TYLCV-IL. Control plants (PepYVMLV DNA-B or mock-agroinoculated) remained asymptomatic.
Higher symptom progression and severity for PepYVMLV DNA-A associated with DNA-B. The kinetics of symptom severity of PepYVMLV DNA-A and TYLCV-IL DNA-A were compared in single or mixed infection with DNA-B (Fig. 2a). Tomato plants agroinoculated with PepYVMLV DNA-A & B exhibited symptoms of leaf crumpling, yellowing and stunting at 12 days post inoculation (dpi) (Fig. 2a). In contrast, tomato plants agroinoculated only with PepYVMLV DNA-A exhibited their first symptoms, which were similar but far less severe, at 27 dpi. Typical symptoms of leaf curling, yellowing and dwarfism were observed in tomato plants agroinoculated with TYLCV-IL DNA-A in single or mixed infection with DNA-B from 12 dpi, although less severe than those induced by PepYVMLV DNA-A & B. Symptom severity increased exponentially, and then linearly, before reaching a plateau with very severe disease symptoms at 22 dpi for PepYVMLV DNA-A coinoculated with DNA-B, and 10 days later for TYLCV-IL in single or mixed agroinfection. The progression of symptom severity (parameter A; i.e. the slope of the linear phase at the inflection point), the time to reach 50% of the maximum severity (parameter B) and the severity at the plateau phase (parameter C) differed significantly between PepYVMLV DNA-A & B and TYLCV-IL DNA-A infection (p = 0.0054, p < 10 -4 and p = 2 × 10 -4 , respectively; Table 2). No significant difference was observed in the virulence kinetics of TYLCV-IL associated or not with DNA-B (p = 0.6, Table 2).
Negative effect of the DNA-B component on tomato growth. Thirty-two days post-agroinoculation, no significant difference in size was observed between the controls and plants inoculated with PepYVMLV DNA-A alone (p = 0.428, Fig. 2b). Conversely, a significant difference in size (p = 0.001) was observed between the control and plants agroinoculated with TYLCV-IL alone. The agroinoculation of PepYVMLV DNA-A and -B to tomato plants strongly affected their growth, with a notable reduction in size compared to plants inoculated with PepYVMLV DNA-A alone (p < 10 -4 , Fig. 2b). In contrast, no significant difference in size was observed between plants agroinoculated with TYLCV-IL DNA-A, in single or mixed inoculation with PepYVMLV DNA-B (p = 0.264).  (Fig. 3a). Experiments were conducted on separate sets    www.nature.com/scientificreports/ alone or in association with DNA-B (Table 3). Based on PCR PepYVMLV DNA-A detection, transmission rates of respectively, 80% and 83% for PepYVMLV DNA-A alone, reached significantly higher values (100%) for PepYVMLV DNA-A and DNA-B in both mixed infection experiments (p = 4 × 10 -5 ). The evaluation of transmission rates based on disease symptoms confirmed the difference between single and mixed infections (p < 10 -4 ), with transmission rates of respectively, 52% and 40% for PepYVMLV DNA-A alone, and respectively, 71% and 69% for PepYVMLV DNA-A and DNA-B together.

Higher number of contaminated phloem parenchyma cells in mixed infection. First, immu-
nofluorescence observations (356 cells out of a total of 49 cross sections), using the nuclear stain DAPI and PepYVMLV-DNA-A & B probes, showed that both DNA-A and DNA-B were exclusively located in the nuclei of phloem parenchyma cells in both single and mixed infections ( Fig. 4a-h). Second, analysis of the images corresponding to mixed infections showed that the two components DNA-A and DNA-B were mostly located together (50-78% of DNA-A and/or -B infected cells, Table 4, Fig. 4e-h). Third, consistent with the greater accumulation of PepYVMLV DNA-A in the presence of DNA-B previously observed in real-time PCR assays, we observed a significantly higher number of contaminated cells in mixed infection (DNA-A and/or -B, p < 0.001), regardless of the sampling date ( Table 4). The number of cells labelled only with DNA-A were similar at 15 and 22 dpi (p > 0.1) but were significantly fewer in mixed infection at 29 dpi (p < 0.001). A minority of cells with only DNA-B were observed in mixed infections (8-14% of infected cells, Fig. 4c,g, and Table 4).

Discussion
Recent studies in Burkina Faso reported the identification of at least five begomovirus species in tomato crops 16,17,22,23 , with the predominance of PepYVMLV. Interestingly, this species of viruses was frequently associated with a previously uncharacterized DNA-B component 15 . To assess whether the DNA-B component is associated with the emergence of PepYVMLV as the currently most prevalent and most severe plant virus disease of tomato crops in Burkina Faso, we evaluated biological traits related to virulence, virus accumulation in the plant and transmission. Agrobacterium-mediated inoculation experiments showed that PepYVMLV DNA-A alone induced systemic and symptomatic infections in N. benthamiana and tomato plants (Fig. 1, Table 1). This result is consistent with PepYVMLV DNA-A genomic organization as a typical Old World monopartite begomovirus 24,25 , and with the role of the V1 ORF in cell-to-cell movement 26 . Such a non-strict association between a monopartite begomovirus and a DNA-B component has been previously described for ToLCGV 27 and TYLCTHV 20 which, in the absence of their cognate DNA-B, are able to induce systemic and symptomatic infections both in the experimental host N. benthamiana 20 and in tomato 27 . In contrast, the New World bipartite begomoviruses absolutely require their cognate DNA-B for systemic movement and for the development of symptoms 5 . Taken together, these experimental results suggest that non-strict bipartite begomoviruses may represent evolutionary intermediates between monopartite and bipartite begomoviruses.
Unlike single agroinoculation of tomato plants with PepYVMLV, mixed agroinoculation with PepYVMLV DNA-A and -B not only strongly increased symptoms of leaf crumpling, yellowing and stunting, as observed in the field, but also led to the death of 17% of inoculated plants under controlled conditions in a climatic chamber. Several studies reported that begomovirus DNA-B contributes to symptom production 27,28 and that BC1 protein is a determinant of pathogenicity 29,30 . This extreme virulence might be a "maladaptive" consequence of the recent association between PepYVMLV DNA-A and DNA-B. If the observed increase in transmission is offset in the long term by a reduced transmission from infected hosts with a shorter lifespan, virulence may decrease in the future 31 . If not, and if coinfections by PepYVMLV DNA-A and DNA-B are frequent, the countries affected this new association would be facing a sort of 'Darwinian Demon' with both high virulence and high transmissibility.
TYLCV-IL has been reported to be one of the most severe and devastating tomato viruses worldwide 32 . In this study, we compared PepYVMLV and TYLCV-IL virulence in association or not with DNA-B. Although when inoculated with TYLCV-IL, some plants also reached maximum severity (score = 9) at 32 dpi, the overall severity score was higher for PepYVMLV associated with DNA-B (earlier appearance of disease symptoms, higher mean symptom score at plateau, higher impact on plant growth). Taken together, these observations demonstrate that PepYVMLV associated with DNA-B is more virulent in tomato in controlled conditions than our TYLCV-IL isolate, and underscores the new global risk of PepYVMLV for the tomato crop if it were to spread beyond West Africa.
The quantification of within-plant accumulation of PepYVMLV DNA-A in single or mixed infection with DNA-B in tomato plants showed that mixed-infected plants contained more PepYVMLV DNA-A than in single infection, and that the copy numbers of the two genomic components presented a positive linear relationship. At the cellular level, FISH analyses of infected plants suggest that this higher viral accumulation is accompanied by a significantly higher proportion of infected phloem cells (DNA-A and/or -B). BV1 and BC1 proteins, encoded by DNA-B which have an analogous function in viral movement to that of the V1/C4 proteins of the monopartite begomoviruses 26 , have been reported to facilitate the escape of some bipartite begomoviruses from the phloem, as well as to infect non-phloem tissues 3 . This has been observed in particular for a strict bipartite geminivirus from the New World, bean dwarf mosaic virus, which is not phloem-limited and was detected in most cell types of the inoculated host leaves 33 . However, in our experiments, PepYVMLV DNA-A, in single or mixed infection with DNA-B, is detected exclusively in the phloem parenchyma of tomato.
In contrast to its significant impact on PepYVMLV infection, DNA-B had no such impact either on TYLCV-IL infection rate or on symptom severity. Interestingly, DNA-B was detected by PCR in only 28% of mixed agroinfected tomato plants, indicating a non-optimal association between TYLCV-IL DNA-A and DNA-B (Table 1). Geminiviruses replicate by a rolling circle mechanism initiated by the binding of the virus-DNA-A encoded replication-associated protein (Rep) 8 www.nature.com/scientificreports/

Conclusion
Our study highlights the role of a DNA-B component in the virulence and transmission of a monopartite begomovirus. The high prevalence and severity of PepYVMLV in tomato crops in Burkina Faso is probably due to the fitness advantage gained through the recruitment of a DNA-B component by the monopartite PepYVMLV. In the case of the non-strict association of PepYVMLV DNA-A and DNA-B, the latter may be regarded as an "extragenomic viral component" that can bolster the pathogenicity, accumulation, and transmission of its cognate virus, while itself depending on the associated virus for successful infection. At the agroecosystem level, when associated with DNA-B, PepYVMLV DNA-A has been recovered from a wide range of hosts 15 , including some weeds that are frequently found in fields. Maintenance of the virus in alternative hosts present in the cultivated area between epidemics enables it to survive the seasonal cycle of tomato cultivation, and may contribute to the predominance of PepYVMLV associated with DNA-B.  40 . Plant size was measured at 32 dpi to assess the effect of viral infection on plant growth. Apical leaves were collected for the detection and the quantification of viral genomes using PCR and real-time PCR, respectively, as described below. Two replicates were performed at different dates for the experiments carried out to assess virulence.

Mechanical inoculation experiments.
Symptomatic tomato leaves were collected from agroinoculated tomato plants, frozen at − 80 °C and ground into a fine powder using a pestle and a mortar with liquid nitrogen, as previously described 41 . Tomato and N. benthamiana seedlings were inoculated by rubbing the leaves with the resulting sap mixed with carborundum powder. All inoculated plants were maintained in the growth conditions described above. Negative controls were mock-inoculated plants. Symptoms were assessed 30 days later, and the plants were tested for the presence of viral DNA by PCR. www.nature.com/scientificreports/ with two successive 50 µL elutions with ultrapure water. Extracts were stored at − 20 °C before use. Conventional PCR was carried out to detect viral DNA in samples collected at 32 dpi and 30 dpi in agroinoculation and whitefly-mediated inoculation experiments, with specific TYLCV-IL 42 and PepYVMLV DNA-A and -B primer sets 15 .

Fluorescence in situ hybridization (FISH). Preparation of the probes. Segments of 85-90 nucleotides
of PepYVMLV DNA-A and DNA-B were amplified by PCR using the GoTaq Polymerase kit (Promega) with specific primers PepYVMLV-A-F/PepYVMLV-A-R and PepYVMLV-B-F/PepYVMLV-B-R according to Ouattara et al. (2020) 15 . PCR products were then migrated in a 1% agarose gel and purified using NucleoSpin® Gel and PCR Clean-up (Macherey Nagel). The resulting amplicons were then used as templates to produce segmentspecific probes using the Invitrogen and Alexa Fluor Bioprime DNA Labelling Kit (Alexa Fluor 488 and 568) as described elsewhere 41 .
Sample preparation and in situ hybridization. Petiole samples from agroinoculated plants were collected at 15, 22 and 29 dpi. Immediately after sampling, the samples were fixed in a phosphate-buffered saline 1 × solution with 4% paraformaldehyde (PFA) and 0.2% Tween-20. Fixed samples were embedded in 8% low melting agarose in a 24-well plate before sectioning with a vibratome (MICROM). Hybridization was performed as previously described 41 .
Microscopy observations. A total of 49 cross sections of tomato petioles were observed by microscopy, comprising 21 and 28 cross-sections from tomatoes infected with PepYVMLV DNA-A in single and mixed infection with DNA-B, respectively. All observations were made using an LSM700 Confocal Microscope (ZEISS) with ZEN software following the protocol of Vernerey et al. 41 . In practice, parameters were adjusted to obtain sufficient resolution and fluorescence intensity signal recovery in a chosen series of infected plant exhibiting a high intensity of fluorescence without saturation points. Images were taken with the 40 × water immersion objective at a resolution of at least 512 × 512 with a pinhole aperture of 1 Airy Unit so as to work in confocal mode. Three sequential tracks were set, one for each fluorochrome used (using lasers at 405 nm for DAPI, 488 nm for Alexa Fluor 488, and 555 nm for Alexa Fluor 568). Analyses were performed using maximum intensity projections so that all the fluorescence emitted by all the nuclei was accounted for. These microscopic images were analysed using Image J software.

Within-plant virus quantification.
Agroinoculated plants sampled at 15, 22 and 29 dpi were used. Each plant sample consisted of five 4-mm-diameter leaf disks collected from the youngest leaves and stored at − 80 °C until analysis. DNA was extracted as described elsewhere 43 . The proportions of PepYVMLV DNA-A and -B were quantified using SYBR Green Real-Time PCR as described by Urbino et al. 43 . Primers were designed and used to quantify the viral molecules in infected plants (for sequences and conditions of use, see Supplementary  table 1). Real-time PCR was performed in a 10-µL reaction mix comprising the 2 × LightCycler® 480 SYBR Green I Master kit (Roche, Germany), each primer, and two microliters of a 1/100 dilution of the DNA template. Plant genomic DNA of each extract was quantified using the nuclear-encoded large subunit ribosomal RNA gene (S. lycopersicum L. 25S ribosomal RNA gene) as described by Conflon et al. 44 . The amplification reactions were run in 384-well optical plates in Roche LightCycler System (Roche, Germany). Amplification conditions were 95 °C for 10 min followed by 40 cycles of 10 s at 95 °C, 30 s at 60 °C and 20 s at 72 °C. Two quantification replicates were performed per sample.
Statistical analysis. All statistical analyses were performed using the R statistical software 45 . Nonlinear regression analyses between the copy number of the DNA-A and DNA-B components were performed, testing different link functions (Cauchy, cloglog, logistic, logit, loglog and probit), to fit the progression of disease severity with gnls function, using the package nmle 46 . Based on the likelihood and using Akaike's information criterion (AIC), the logit function was selected as the best model. In this model, written y ~ 1 + C/(1 + exp(− A * (x − B))), the disease severity (y) is dependent on the dpi (x) and three biologically relevant parameters where A is the slope of the linear phase at the inflection point, 1 + C is the disease severity at the plateau phase, and B is the time to reach 50% of disease severity at the plateau phase. The estimated parameters were then compared between the different conditions using likelihood ratio tests in nested models. For these analyses, only plants were used for which single (DNA-A) or mixed (DNA-A and -B) infections were validated by PCR. Real-time PCR data were expressed as the log of the ratio of the quantity of virus DNA to that of plant DNA. The amount of the DNA-A component in single and mixed inoculation conditions was compared using an ANOVA F-test. For mixed infections, linear regression was used to estimate the correlation between the copy numbers of DNA-A and DNA-B component copy numbers. FISH data were subjected to multiple comparisons of means using post-ANOVA Tukey HSD-test 47 .
Ethical statement. All the experimental protocols involving plants adhered to relevant ethical parameters/ regulations.